Technology to detect minute quantities of nucleic acids has advanced rapidly over the last two decades including the development of highly sophisticated amplification techniques such as polymerase chain reaction (PCR). Researchers have readily recognized the value of such technology to detect nucleic acids which are indicative of diseases and genetic features in human or animal test specimens. The use of probes and primers in such technology is based upon the concept of complementarity, that is, the bonding of two strands of a nucleic acid by hydrogen bonds between complementary nucleotides (also known as nucleotide pairs).
PCR is a significant advance in the art to allow detection of very small concentrations of a targeted nucleic acid. The details of PCR are described, for example, in U.S. Pat. No. 4,683,195 (Mullis et al), U.S. Pat. No. 4,683,202 (Mullis) and U.S. Pat. No. 4,965,188 (Mullis et al), although there is a rapidly expanding volume of literature in this field.
In order to effectively amplify and detect a target nucleic acid, it is usually necessary to isolate that nucleic acid from cellular and other specimen debris. Various lysing procedures are known, including freezing, treatment with digesting enzyme such as proteases (for example, Proteinase K), boiling, and use of various detergents (see for example U.S. Ser. No. 178,202, filed Apr. 6, 1988 by Higuchi, and EP-A-0 428 197, published May 22, 1991), solvent precipitations and heating protocols.
Circulating DNA has been detected in blood serum and plasma. Nanogram quantities are detected in normal subjects (Steinman, C. R., J Clin. Invest. 56:512-515, 1975 and Raptis, L., et al., J. Clin. Invest. 66:1391-1399, 1980), and increased levels are detected in chronic autoimmune diseases (Leon, S. A., et al., Cancer Res., 37:646-650, 1977) and in cancer patients (Stroun, M., et al., Eur. J. Cancer Clin. Oncol. 28:707-712, 1987; Maebo, A., Jpn. J. Thorac. Dis. 28:1085-1091, 1990; Fournie, G. J., et al., Cancer Lett., 91:221-227, 1995; Lin, A., et al., BioTechniques 24:(6) 937-940, 1998; and Sorenson, G. D., et l., Cancer Epidemiology, Biomarkers and Prevention 3:67-71., 1994). Recently, it has become evident that free extracellular DNA present in blood serum and plasma can be used for genotype analysis (Lin, A., et al., BioTechniques 24:(6) 937-940, 1998), for detection of cancer (Mulcahy, H. E., et al., Clin. Cancer Res. 4:271-275, 1998), and DNA in maternal serum may be used in prenatal diagnostics (Lo Dennis, et al., Am. J. Human Genet. 62:768-775, 1998). Mutations present in a primary tumor, often can be detected using DNA from blood plasma or serum DNA (Sorenson, G. D., et al., Cancer Epidemiology, Biomarkers and Prevention 3:67-71., 1994; Vasyukhin, V., et al., In Challenges of Modern Medicine, Vol. 5, Biotechnology Today, R. Verna, and A. Shamoo, eds, 141-150. Aera-Serono Symposia Publications, Rome; Mulcahy, H. E., et al., supra.; Kopreski, M. S., et al., Brit. J. Cancer 76:1293-1299, 1997; Chen, X., et al. Nature Medicine 2: 1033-1035, 1996; Vasioukin, V., et al., Brit. J. Haematology 86:774-779, 1994; and Tada, M., et al., Cancer Res. 53:2472-2474, 1993). Thus, DNA present in serum and plasma represents a minimally invasive source for information related to cancer diagnosis, prognosis, and therapy.
To effectively amplify and detect a target nucleic acid, it is usually necessary to separate the nucleic acid from interfering substances present in a specimen of interest. Several different approaches have been used to concentrate and purify DNA from blood serum or plasma. Many of these methods involve multiple steps including phenol, ether, and chloroform treatment, dialysis, passage through Concanavalin A-Sepharose to remove polysaccharides and then centrifugation in a cesium chloride gradient (Vasyukhin, V., et al., supra.). More recently, Qiagen has commercialized a system for DNA concentration and purification based on a spin column protocol. The Quiagen protocol is complex, involving a total of eight steps, treatment with a protease, incubations at 70° C., and requires the use of at least 3 different buffers, in addition to a silica spin column centrifugation step.
Recently, Goecke et al. (WO 97/34015) reported the detection of extracellular tumor-associated nucleic acid in blood plasma and serum using nucleic acid amplification assays. In their preferred method, DNA is co-precipitated from plasma and serum using a multistep protocol involving an initial co-precipitation by gelatin, followed by solvent treatment and centrifugation. Other time-consuming and complex protocols involving the use of glass beads, silica particles or diatomaceous earth for extraction of DNA from serum and plasma are also described.
The use of weakly basic polymers for the capture and selective release of nucleic acids has been described U.S. Pat. No. 5,622,822 (Ekeze et al.), U.S. Pat. No. 5,582,988 (Backus et al.), and U.S. Pat. No. 5,434,270 (Ponticello, et al.). The protocols described in the aforementioned patents depend upon the use of a cell lysing agent or a cell lysing step. Surfactants are often used as cell lysing agents. The use of surfactants and other lysing agents results in the release of nucleic acids from cells and cellular components in blood; causing a large concentration of background DNA.